1. Field of the Invention
The ability to synthesize polynucleotide fragments having a desired nucleotide sequence is a useful tool in both research and applied molecular biology. Short synthetic polynucleotides (oligonucleotides) are useful as adaptors or linkers in joining longer DNA segments, and as hybridization probes and DNA synthesis primers. Longer polynucleotides can be constructed from shorter segments having overlapping cohesive ends and used as structural genes, regulatory regions such as promoters, terminators, operators, and the like. It is thus of great interest to provide convenient automatic techniques for producing synthetic DNA fragments with high yields in a relatively short time.
At present, a variety of approaches for polynucleotide synthesis are available. These approaches can be characterized based on several criteria. First, the synthesis is usually carried out either on a solid-phase substrate or in solution. Solid-phase synthesis relies on sequential addition of mononucleotides to a growing chain attached at one end to the substrate. The solid-phase allows easy separation of the reactants, but the method requires excess quantities of reactants and usually provides only small quantities (less than 1 mg) of the desired sequence. Solution phase synthesis, while it requires lesser amounts of the expensive reagents and can provide larger quantities of the product sequence, requires isolation and purification of the intermediate product after every addition. Virtually all automated polynucleotide systems rely on solid-phase synthesis.
Second, the synthesis chemistry must be selected. There are presently two chemistries in widespread use for automated polynucleotide synthesis. The triester method, as described by Catlin and Cramer (1973) J. Org. Chem. 38:245-250 and Itakura et al. (1973) Can. J. Chem. 51:3649-3651, relies on the addition of suitably blocked phosphate-triester intermediates which are generally inexpensive and stable. The phosphite-triester method, as described by Letsinger and Lunsford (1975) J. Am. Chem. Soc. 98:3655, is somewhat more complex, but generally provides higher yields than the phosphate-triester method. The utility of the phosphite-triester method was greatly improved by the use of N,N-dialkylamino phosphites (amidites) which are more stable than the phosphorochlorodite intermediates initially employed. While the phosphite-triester method is often favored because of the greater yield at each nucleotide addition, the phosphate-triester method is also suitable for automated polynucleotide synthesis.
The third choice which must be made is the selection of a reactor system. Heretofore, solid-phase reactor systems have generally employed either (1) a tight bed column, (2) a loose bed column, or (3) a batch reactor. The tight bed column is tightly packed with the solid-phase support and the reactants are introduced either in a single pass of by a recirculating stream. Generally, such tight pack columns inhibit the mass transfer of reagents and the resulting slow diffusion rates for the reactants into the support increase the necessary reaction time and/or decrease the efficient utilization of reactants. A second problem experienced with such tight bed columns is channeling through the column packing which aggravates the mass transfer problems just discussed.
To partially alleviate these problems, loose bed columns (containing a much higher void volume than the tight bed columns) have been introduced. By slowly passing the reactant through the column, higher mass transfer rates are achieved and utilization of the expensive reactants is improved. Also, channeling is reduced since the solid phase packing will shift to equalize the flow profile therethrough. Although an improvement, the resulting mass transfer is still limited by the lack of agitation within the reactor. Moreover, the shifting of the solid phase can generate very fine particles which can plug the frits used to contain the solid phase within the reactor. This problem is also experienced by the tight bed columns, although to a lesser extent.
In a batch reactor, the support matrix is held in an enclosed vessel. Reactants are introduced and the vessel contents agitated, typically by bubbling an inert gas through the liquid in the reactor. While such a system can provide very efficient utilization of the reactants (by increasing the retention time in the reactor), relatively large volumes of the reactant and solvents are necessary to fill the reactor. Moreover, the mixing in the batch system is typically not complete, and the solid-phase support is often deposited on the vessel wall above the reagent level so that it is no longer exposed to the incoming reagents.
In addition to the above limitations, all three of the reactor systems just described suffer from bubble formation on the solid-phase substrate which inhibits the reactions and causes lower yields. The bubble formation is caused by certain solvents (in particular the methylene dichloride) which de-gas on the solid-phase substrate.
In view of the above, it would be desirable to provide a system and method for polynucleotide synthesis which would avoid or reduce the shortcomings of the prior art systems and allow the efficient utilization of process reactants with relatively short retention times for each reactant. In particular, it would be desirable to provide a reactor which affords thorough mixing of reactants to increase the mass transfer rate and which remains free from plugging.
2. Description of the Prior Art
Methods and apparatus for the automated solid-phase synthesis of polynucleotides are described in U.S. Pat. No. 4,373,071 to Itakura; European Patent Application No. 81101449.5; and in Alvarado-Urbina et al. (1981) Science 214:270-274. Automated systems for the synthesis of polynucleotides are available from Biosearch, San Rafael, California; Bio Logicals, Toronto, Canada; Applied Biosystems, Foster City, California; and Bethesda Research Laboratories, Inc., Gaithersburg, Maryland. These systems and others generally rely on the synthesis techniques described above.